Bacterial plasmids are double-stranded closed circular DNA molecules that range in size from 1 kb to more than 200 kb. They are found in a variety of bacterial species, where they behave as accessory genetic units that replicate and are inherited independently of the bacterial chromosome. Nevertheless, they rely on enzymes and proteins encoded by the host for their replication and transcription. Frequently, plasmids contain genes coding for enzymes that under certain circumstances in nature are advantageous to the bacterial host. Among the phenotypes conferred by plasmids are resistance to antibiotics; production of colicins and enterotoxins; and restriction and modification enzymes.
Plasmids are useful tools in genetic engineering. They can be joined with fragments of foreign DNA in vitro to form chimeras that can be introduced into bacterial host cells; amplified and isolated or expressed (See for example, U.S. Pat. Nos. 4,237,234; 4,740,470 and 4,468,464 to Cohen et al.). A variety of plasmids have been developed to perform specialized functions. For example, plasmids have been constructed with powerful promoters to generate large amounts of mRNA complementary to cloned sequences of foreign DNA and thereby express high levels of protein.
Various plasmids (e.g. pUC), cosmids and phagemids (e.g. pEMBL, pGEMA) are useful cloning vectors for initiating large scale sequencing projects (T. Maniatis, E. F. Fritsch and J. Sambrook (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Methods in Enzymology, Vol. 101 (1983), Recombinant DNA, Part C; Vol. 153 (1987), Recombinant DNA, Part D; Vol. 154 (1987), Recombinant DNA, Part E; Vol. 155 (1987), Recombinant DNA, Part F and Vol. 152 (1987), Guide to Molecular Cloning Techniques, Academic Press, New York). These vectors accomodate cDNA or genomic libraries of large DNA fragments.
However, the large DNA fragments generated typically can not be sequenced directly in one run, since Sanger sequencing chemistry only allows about 200 to 500 bases to be read at a time. As a result, long DNA fragments typically must be cut into shorter pieces which are separately sequenced. In one approach this is done in a fully random manner by using, for example, unspecific DNAse I digestion, frequently cutting restriction enzymes, or sonification, and sorting by electrophoresis on agarose gels (Methods in Enzymology, supra). However, this method is time-consuming and often not economical as several sequences are sequenced many times until a contiguous DNA sequence is obtained. Very often the expenditure of work to close the gaps of the total sequence is enormous.
Several strategies have been proposed for sequencing long DNA fragments in a non-random, i.e. direct, way from one end through to the other (Methods of Enzymology, supra; S. Henikoff, Gene, 28, 351-59 (1984); S. Henikoff, et al. U.S. Pat. No. 4,843,003; and PCT/Application WO 91/12341). However, none of these sequencing methods provide an acceptable method of sequencing megabase DNA sequences in either a timely or economical manner. The main reason is that these methods all rely on polyacrylamide gel electrophoresis (PAGE) as a central and key element of the overall process.
PCT patent application international publication number WO 94/16101 by Koster describes DNA sequencing procedures, which utilize Sanger base-specific, chain termination reactions to generate from an unknown DNA molecule, nested fragments that are analyzed by mass spectrometry to determine the sequence of the unknown DNA, PCT patent application international publication number WO 94/21822 by Koster describes sequencing techniques in which the mass of remaining nucleic acid molecules or the nucleotides sequentially cleaved by an exonuclease activity are analyzed by mass spectrometry to identify the unknown nucleic acid molecule.
Although restriction analyses provide useful information on an unknown DNA sequence, hybridization screening methods can reveal whether a particular sequence is present in an unknown DNA sequence and high throughput sequencing methods can provide the exact sequence of the unknown DNA, the potential of these methodologies have not as yet been realized. One problem is that existing procedures for isolating plasmids from bacterial cells are hampered by centrifugation steps and the processing of single reaction tubes. Centrifugation, which is used to collect cells, remove cellular debris and yield the DNA by ethanol precipitation, can be done simultaneously only with a small number of samples. The handling of single reaction tubes is time-consuming and bears the risk of misplacing samples. To circumvent these problems, methods for the purification of bacteriophage M13 sequencing templates in 96-well microtiter (mt) plates have been developed (Smith, V. et al., (1990) DNA Sequence 1, 73-78; and Alderton, R. P. et al., (1992) Anal. Biochem. 201, 166-169). Microtiter filter plates have also been employed for harvesting M13 from culture supernatants (Eperon, I. C. (1986) Anal. Biochem. 156, 406-412).
A means to rapidly isolate large numbers of plasmid DNA from plasmid containing cells is needed, particularly for screening large numbers of clones (e.g. for mutations), performing restriction analyses or for performing high throughput DNA sequencing.